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. 2004 May;136(2):388–392. doi: 10.1111/j.1365-2249.2004.02427.x

Detection of circulating soluble CD28 in patients with systemic lupus erythematosus, primary Sjögren's syndrome and systemic sclerosis

M HEBBAR *, P JEANNIN †, G MAGISTRELLI †, P-Y HATRON *, E HACHULLA *, B DEVULDER *, J-Y BONNEFOY †, Y DELNESTE †
PMCID: PMC1809021  PMID: 15086406

Abstract

The aim of this study was to evaluate the presence and the role of the serum soluble costimulatory molecule CD28 in patients with systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), and systemic sclerosis (SSc). Soluble CD28 concentration was determined by ELISA in 45 patients with SLE, 45 patients with primary SS, 30 patients with SSc, and 45 healthy subjects. We also evaluated CD28 mRNA expression by semiquantitative RT-PCR, and the biological activity of recombinant soluble CD28 on T lymphocyte activity. Concentrations of soluble CD28 were significantly higher in patients with SLE, primary SS and SSc than in healthy subjects. Soluble CD28 concentrations were higher in patients with systemic primary SS than in patients with glandular-limited primary SS. PCR analysis suggested that soluble CD28 resulted from the shedding of the membrane form. In vitro assay revealed that soluble CD28 inhibits the anti-CD3 mAb induced T cell proliferation. Soluble CD28, which modulates the proliferation of T lymphocytes, could be associated with disease severity in patients with autoimmune disease, especially primary SS. These results suggest that soluble CD28 could play an important role in the regulation of autoimmune diseases.

Keywords: soluble CD28, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis

INTRODUCTION

The initiation of T cell activation requires a primary signal delivered by the antigenic peptide presented by MHC molecules and a non specific signal generated by the interaction of costimulatory molecules [1,2]. The costimulatory signal (also called second signal) results from the interaction of molecules belonging to the CD28 family expressed by T cells, and molecules belonging to the B7 family expressed by the antigen presenting cells. CD28 is constitutively expressed by T cells [3] and interacts with the molecules B7-1 (CD80) [4] and B7-2 (CD86) [5]. This interaction is required to induce a functional T cell activation (induction of IL-2 production and clonal expansion). Blocking the CD28/B7 interaction results in anergy [6] or apoptosis [7]. The important role played by the interaction of CD28 with B7 molecules in the generation of an antigen-specific T cell response is illustrated in CD28 deficient mice that exhibit reduced Th responses and Ig class switching [8]. More recently, the B7 family has been extended with the identification of B7-RP1 [9,10] and B7-H3 [11] which provide an activation signal, and PD-L1 and PD-L2 which inhibit T cell proliferation [12,13]. ICOS, a CD28-like molecule expressed on activated and resting memory T cells and induced on naive T cells [14,15], binds to B7-RP1. PD1 is a counter-structure for PD-L1 and PD-L2 [12]. Nevertheless, among these costimulatory molecules, it is recognized that CD28 plays a primordial role for the priming of naive T cells [16]. More recently, soluble costimulatory molecules have been reported, increasing the complexity of T cell behaviour [17–20].

Autoimmune diseases are associated with impaired T cell responses and a high percentage of peripheral blood T cells is activated in patients. A defective T cell proliferative response was observed in systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS) [21–23]. Studies have reported abnormal costimulatory molecule expression on peripheral blood and infiltrating T cells in patients with autoimmune diseases. The expression of CD80 and CD86 is increased on circulating T cells in patients with SLE, but not in rheumatoid arthritis (RA) and primary SS [24] and CD80 was detected on synovial T cells in RA patients [25]. The frequency of CD28 negative T cells is increased in RA, SLE, and Wegener's granulomatosis [21, 26, 27]. The potential role of the costimulatory molecules in the generation and regulation of autoimmune diseases was evidenced in mouse models: spontaneous autoimmunity [28,29] and experimentally induced autoimmune encephalomyelitis [30] or collagen-induced arthritis [31] are prevented by CTLA-4-Ig or combinations of neutralizing anti-CD80 and anti-CD86 mAbs. Dysregulation in soluble costimulatory molecule production was also evidenced in autoimmune diseases: soluble CD80 was detected in synovial fluid of arthritis patients [32] and soluble CTLA-4 was found in the serum of patients with autoimmune thyroid disease [19].

Based on the critical role played by CD28 in the immune response, the aim of this study was to evaluate whether soluble CD28 could be detected in patients with SLE, primary SS and systemic sclerosis (SSc). The functional activity of soluble CD28 was also analysed.

PATIENTS AND METHODS

Patients

This study was performed on:

  • 45 patients with SLE fulfilling the American College of Rheumatology (ACR) criteria [33], sex ratio (M/F): 6/39, mean age 32·1 ± 18·7 (range 16–59) years, disease duration 3·4 ± 2·5 (range 0–7) years. Disease activity was evaluated using the SLE Disease Activity Index (SLEDAI) [34].

  • 45 patients with primary SS fulfilling the European criteria [35], sex ratio: 3/42, mean age 50·5 ± 13·8 (range 17–78) years, disease duration 4·4 ± 4·1 (0–18) years. Patients were divided into two groups: group 1: glandular-limited SS (isolated sicca syndrome), 14 patients; group 2: systemic SS (presence of extraglandular manifestations: arthritis, vasculitis, lymph node enlargement, visceral envolvement) – 31 patients.

  • 30 patients with SSc fulfilling the ACR criteria [36]: sex ratio: 7/23, mean age 54·1 ± 14·6 (range 22–86) years, disease duration 4·9 ± 5·3 (range 0–20) years. Extent of sclerosis was graded according to Barnett's [37] and Coventry's classification system: grade I (sclerodactyly – 19 patients), grade II (skin stiffness including the metacarpophalangeal joints but sparing the trunk – 7 patients), and grade III (diffuse skin stiffness inluding the trunk – 4 patients). Nineteen patients had an isolated SSc. Eleven patients had a SSc associated with features of other systemic diseases: presence of a secondary SS in 7 cases, features of SLE and/or polymyositis (mixed connective tissue disease) in 4 cases.

  • 45 healthy subjects, sex ratio: 10/35, mean age 48·1 years ± 11·7 (range 20–62).

All these caucasian patients were chosen randomly. All had a recently diagnosed disease, and none of them received immunomodulatory therapy. A laboratory analysis was performed in all patients to determine levels of serum gammaglobulin and creatinine, and to test for antinuclear antibodies (indirect immunofluorescence technique), anti-SS-A and anti-SS-B Abs (Ouchterlony double diffusion in agarose gel), antinative DNA antibodies (radio-immunoassay), and hypocomplementaemia.

Detection of soluble CD28 by ELISA

An immunoreactive form of CD28 was evaluated in human serum by ELISA. Briefly, the anti-CD28 mAb (clone CD28·2) (BD Biosciences, Palo Alto, CA, USA) was coated (2 ng/100 µl/well) in 96 well plates (Nunc, Oslo, Norway) in 0·1 m phosphate buffer pH 4·0 (16 h at 4°C) before incubation for 2 h at room temperature with PBS/BSA 1%. After washing, plates were incubated for 16 h at 4°C with undiluted serum (200 µl/well). After washings, bound CD28 was detected with a biotin labelled anti-CD28 polyclonal Ab (R & D Systems, Abingdon, UK), followed by incubation with streptavidin-biotinylated HRP (used at 1/5000; Amersham Biosciences, Uppsala, Sweden) revealed with the substrate o-phenylene diamine (Sigma, St Louis, MO, USA). The specificity was determined using recombinant soluble CD28 and, as negative controls, soluble CD86 [20], soluble CTLA-4-Fc (Ancell, Bayport, MN, USA) and human cytokines (all from R & D Systems). Results are expressed in ng/ml, mean ± sd.

Analysis of CD28 mRNA expression

The expression of the mRNA encoding CD28 was determined by RT-PCR. Briefly, cytoplasmic RNA was extracted from freshly isolated PBMC using the RNeasy kit (Qiagen, Courtaboeuf, France) following the manufacturer's recommendations. The single-strand cDNA was synthesized using 2 µg of total RNA by reverse transcription using an oligo-dT primer (Amersham Biosciences). PCR reactions were performed with cDNA corresponding to 10 ng of total RNA, and primers designed to amplify the entire coding sequence of CD28 [17]. PCR reaction was as follows: 94°C for 5 min, 30 cycles 94°C for 1 min, 60°C for 1 min and 72°C for 1 min followed by a final extension at 72°C for 5 min. RNA integrity and cDNA synthesis was verified by amplifying GAPDH cDNA. The amplified fragments were size-separated on a 1% agarose gel and visualized by ethidium bromide.

Functional activity of recombinant soluble CD28 on human T cells

Production of recombinant soluble CD28.

The cloning of CD28a cDNA, a spliced variant of CD28 with a deletion of exon 3 resulting in the loss of the transmembrane domain, has been previously reported [17]. The CD28a cDNA was subcloned in the pCDNA3·1 vector (Invitrogen, Groningen, the Netherlands) in frame with a c-myc encoding sequence and used to transfect COS-7 cells (ATCC, Manassas, VA, USA). COS-7 cells were cultured in DMEM medium supplemented with 10% FCS, l-glutamine and antibiotics (all from Life technologies) and transfected by lipofection (Fugene™ 6; Boehringer Mannheim, Mannheim, Germany). Soluble recombinant CD28 was produced in serum free medium (OptiMEM; Life technologies) and purifed using immobilized antic-myc Ab.

In vitro proliferation assay and IL-2 quantification.

Human PBMC were cultured in 96-well culture plates (Nunc) (2·5 × 105 cells/ml, 200 µl/well) and were either non stimulated or stimulated with 30–100 pg/ml anti-CD3 mAb (clone OKT3) in the absence or presence of 10 ng/ml anti-CD28 mAbs or 0·01–1 µg/ml purified soluble CD28. In some experiments, recombinant soluble CTLA4-Fc (Ancell, Bayport, MN, USA) was used as a control. After 5 days, cells were pulsed with 0·25 µCi/well 3H-thymidine (Amersham Biosciences) for 6 h. Radioactive incorporation was measured by standard liquid scintillation counting. Results are given in index of proliferation defined as the ratio A/O where A and O are the cpm values in activated and non activated cells, respectively. IL-2 was quantified by ELISA in the 48 h culture supernatants, following the manufacturer's recommendations (R & D Systems).

Statistical analysis

Mann–Whitney test was used to compare mean soluble CD28 concentrations. Linear regression test was used to compare soluble CD28 concentrations and SLEDAI.

RESULTS

Serum soluble CD28 concentrations

Soluble CD28 was detected in the serum of patients with SLE (132 ± 353 ng/ml, mean ± SEM), primary SS (290 ± 504 ng/ml), and SSc (83·3 ± 251 ng/ml) (Fig. 1). Levels of soluble CD28 in healthy subjects were 38·75 ± 82·4 ng/ml (Fig. 1). Concentrations of soluble CD28 found in patients with SLE, primary SS and SSc were significantly higher than those found in the group of healthy subjects (P < 10−2, P < 10−4, P < 0·05, respectively). The mean soluble CD28 concentration was significantly higher in patients with primary SS than in patients with SSc (P < 0·05). In patients with SLE, the mean SLEDAI was 8·8 ± 2·3. No correlation was found between soluble CD28 concentrations and SLEDAI. Two patients with SLA had very high soluble CD28 levels (>1000 ng/ml). These two patients had and active SLE: one with pleuropericarditis, one with glomerular involvement. Patients with systemic primary SS had significantly higher soluble CD28 levels than patients with glandular-limited primary SS (465 ± 810 versus 28 ± 35 ng/ml; P < 10−2). Seven patients with primary SS had very high soluble CD28 levels. Five of them had a systemic disease, three a cutaneous vasculitis, and two diffuse nodal involvement (but without evidence for lymphoma). Patients with SSc associated with secondary SS or mixed connective tissue disease had significantly higher soluble CD28 levels than patients with isolated SSc (84 ± 0·23 versus 1·3 ± 1·8 ng/ml; P < 0·05). No correlation was found between soluble CD28 levels and extent of sclerosis in SSc. No immunoreactivity was observed with soluble CD86, soluble CTLA-4-Fc and all the cytokines tested (data not shown).

Fig. 1.

Fig. 1

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Detection of soluble CD28 in human serum. Soluble CD28 was detected by ELISA in serum from systemic lupus erythematosus (SLE) (n = 45), primary Sjögren's syndrome (SS) (n = 45), and systemic sclerosis (SSc) (n = 30) patients, and healthy subjects (n = 45).

In all patient groups, no correlation was found between soluble CD28 concentrations and biological features (gammaglobulin and creatinine levels, antinuclear antibodies, and hypocomplementaemia).

Analysis of CD28 mRNA expression

We previously reported the expression of alternatively spliced CD28 mRNA variants in non stimulated T cells from healthy subjects [17]. We then analysed whether CD28 mRNA expression could be affected in T cells from patients. Only the full length transcript (i.e. encoding membrane CD28) was expressed in T cells from five patients (selected on high levels of circulating CD28) (Fig. 2); the lack of splicing was confirmed by sequencing the PCR fragment (data not shown). Conversely, three mRNA variants were expressed in T lymphocytes from healthy subjects, as previously reported (Fig. 2) [17]. These results suggested that soluble CD28 in patients could be generated by shedding of the membrane form rather than transcription of an alternatively spliced mRNA.

Fig. 2.

Fig. 2

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CD28 mRNA expression in patients. The expression of CD28 transcripts was evaluated by RT-PCR in 5 patients (P1-P5) exhibiting the higher levels of soluble CD28 (irrespective of the pathology) and in 5 healthy subjects (representative result from only one subject are presented). RNA integrity and cDNA synthesis was verified by amplifying GAPDH mRNA.

Functional activity of soluble CD28 on T cell activation

A recombinant c-myc tagged form of soluble CD28 was produced and used in in vitro assays. Stimulation of PBMC from healthy subjects with anti-CD3 mAb or anti-CD3 plus anti-CD28 mAbs induced a potent and dose-dependent T cell proliferation (mean proliferation index (±SD); 9 ± 1 and 62 ± 9, respectively) (Fig. 3). Soluble CD28 inhibited in a dose-dependent manner the anti-CD3 mAb-induced T cell proliferation (76 ± 12% inhibition with 1 µg/ml soluble CD28) (Fig. 3). As control, 1 µg/ml soluble CTLA4-Fc also inhibited the proliferation of anti-CD3 mAb stimulated T cells (95 ± 8% inhibition) (Fig. 3).

Fig. 3.

Fig. 3

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Soluble CD28 inhibits T cell proliferation. PBMC from healthy subjects were activated with anti-CD3 mAb (αCD3) without or with 0·1–10 µg/ml soluble recombiannt CD28 or 10 µg/ml soluble CD28-Fc. Control was proliferation induced by anti-CD3 plus anti-CD28 mAbs (αCD3 plus αCD28). Results are expressed in proliferation index (mean ± SD; n = 5).

DISCUSSION

In the present study, we report the detection of soluble CD28 in the serum of patients with SLE, primary SS and SSc. The concentrations of soluble CD28 were significantly increased in these patients compared with healthy subjects. In patients with SLE, levels of soluble CD28 did not correlate with disease activity (as assessed by the SLEDAI). However, the SLEDAI scoring system has been performed to be used in clinical routine, and the lifethreatening items (i.e. nervous involvement) have higher weights than the biological markers, suggesting that the biological disease activity is not accurately evaluated by this scoring system. Native anti-DNA Abs and complement activation are the biological markers routinely used to evaluate disease activity in SLE patients. Several studies have shown that these markers are not sufficiently reliable [38,39]. So, it would be of interest to assess the value of soluble CD28 in a prospective longitudinal study in a large series of patients with SLE, with regard to the respective value of the biological markers, soluble CD28, native anti-DNA Abs and hypocomplementemia.

No established disease activity scoring system has been proposed in patients with primary SS. In the present study, the patients have then been divided into two groups based on the clinical manifestations: patients with primary SS limited to glandular involvement and patients with systemic manifestations. The soluble CD28 concentrations were significantly higher in patients with systemic primary SS.

SSc is a connective tissue disease mainly characterized by collagen accumulation in skin and internal organs, and by endothelial cell activation. In some patients, clinical and biological features suggest an immunological involvement (association with an autoimmune disease, presence of antinuclear Abs). In the present study, we found that SSc patients exhibiting high levels of circulating soluble CD28 generally had an associated Sjögren's syndrome or features of other autoimmune diseases (SLE and/or polymyositis). So, these results do not bring evidence for a role of immunological abnormalities in pathogenesis of isolated SSc. We and others have suggested a prominent role of endothelial and mast cells [40,41]. Taken together, these data suggest that soluble CD28 can be detected in the serum of patients with autoimmune disease, and especially Sjögren's syndrome. Moreover, the results suggest that the soluble CD28 concentrations could be associated with the disease severity. Of note is the fact that we also measured the concentrations of soluble CTLA-4 and soluble CD86 in all these patients. Concentrations were not significantly different than those found in healthy subjects (data not shown).

Soluble CD28 can be produced either by shedding of the membrane form or can result from alternative mRNA splicing, as previously reported [17]. RT-PCR analysis showed that only the full length CD28 transcript (i.e. encoding membrane CD28) was detected in peripheral blood cells from patients exhibiting soluble CD28, suggesting that soluble CD28 found in patients could result from a shedding of the membrane form. In accordance with this result, analysis of membrane CD28 expression on T cells from a limited number of patients with primary SS who had a high soluble CD28 level showed a reduced expression compared with healthy subjects (data not shown). Purification and sequencing of circulating soluble CD28 are in progress to respond to this question. Moreover, the fact that exclusive full length CD28 expression is detected in activated T lymphocytes [17] confirms that peripheral T cells in these patients are in an activated state, as previously reported [24–27]. Several studies have shown that the proliferative T cell response is altered in some patients with autoimmune diseases [42]. A decreased autologous mixed lymphocyte reaction has been reported in patients with SLE, primary SS, and SSc [21, 43, 44]. The mitogenic- and anti-CD3 mab-induced T cell proliferation was reduced in patients with primary SS [22]. An altered T cell proliferation associated with the presence of soluble CD28 prompted us to suspect an involvement of soluble CD28 in this activity. In vitro experiments showed that recombinant soluble CD28 prevented T cell proliferation induced by the anti-CD3 mAb or by mitogens. These results are in agreement with previous studies reporting that blocking costimulatory molecules prevented anti-CD3 mAb-induced T cell activation. Taken together, these data suggest that soluble CD28 could be involved, at least in part, in the T cell hyporesponsiveness observed in these patients.

To conclude, we show that soluble CD28 concentrations are frequently increased in patients with systemic autoimmune disaeses, and that soluble CD28 inhibits T cell response in vitro. The value of soluble CD28 concentrations measurement in evaluating disease activity should be assessed.

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